PROJECT SUMMARY AND ABSTRACT Cytochromes P450 (CYPs) constitute a wide-ranging class of heme containing enzymes that mediate catalysis for a diverse array of reactions. A common feature for all CYP mediated reactions is reliance on accessory proteins as electron donors, allosteric modulators, or both. The small heme protein cytochrome b5, for example, is a well-characterized modulator of microsomal CYP activity. However, the regulatory scheme for the P450 systems in the mitochondria (where b5 is not available as a modulator) is not currently understood, thereby representing a fundamental gap in the knowledge of how these systems work. Therefore, the overarching goal of this research program is to outline the structural framework for how the activity of mitochondrial CYPs enzymes are regulated. As an important first step toward this goal, this proposal outlines a structure and function study for two vitamin D metabolizing mitochondrial enzymes, CYP27B1 and CYP24A1. While CYP27B1 activates vitamin D via a 1?-hydroxylation of the vitamin D pre-hormone, CYP24A1 exerts the opposite effect by deactivating via a carbon 23 or carbon 24 hydroxylation of the vitamin D side chain. In concert, they exhibit tightly regulatory control that governs vitamin D bioavailability. The enclosed preliminary data demonstrates that formation of the CYP-Adx electron transfer complex produces a long-range change in CYP24A1 structure that perturbs the enzyme's ability to bind vitamin D and that is also consistent with a closing off of the CYP active site. These data form the basis for the overall working hypothesis that substrate binding and electron transfer (steps 1 and 2 in CYP catalysis) participate in an allosteric regulation of CYP function in mitochondria. Over the next five years, the Estrada lab plans to test elements of this hypothesis as a set of project goals that utilize a multidisciplinary tool kit including, among other techniques, nuclear magnetic resonance spectroscopy, X-ray crystallography, chemical cross-linking, molecular dynamics simulations, and functional assays. The goal of this effort is to address fundamental questions pertaining to mitochondrial CYP function, structure, and regulation of function. While the work proposed here outlines the short-term goals of this research program, completing these goals will lay the foundation for the study of additional mitochondrial CYPs, leading to a sustainable long-term research program.